Ampholyte Displacement Research Articles

Method for Analyte Identification Using Isotachophoresis and a Fluorescent Carrier Ampholyte Assay

We present a novel method for identification of unlabeled analytes using fluorescent carrier ampholytes and isotachophoresis (ITP). The method is based on previous work where we showed that the ITP displacement of carrier ampholytes can be used for detection of unlabeled (nonfluorescent) analytes. We here propose a signal analysis method based on integration of the associated fluorescent signal. We define a normalized signal integral which is equivalent to an accurate measure of the amount of carrier ampholytes which are focused between the leading electrolyte and the analyte. We show that this parameter can be related directly to analyte effective mobility. Using several well characterized analytes, we construct calibration curves relating effective mobility and carrier ampholyte displacement at two different leading electrolyte (LE) buffers. On the basis of these calibration curves, we demonstrate the extraction of fully ionized mobility and dissociation constant of 2-nitrophenol and 2,4,6-trichlorophenol from ITP experiments with fluorescent carrier ampholytes. This extraction is based on no a priori assumptions or knowledge of these two toxic chemicals. This technique allows simultaneous identification of multiple analytes by their physiochemical properties in a few minutes and with no sample preparation.

Purification and properties of β- n-acetylhexosaminidase a from pig brain

1. 1. Adult pig brain β- N-acetylhexosaminidase was separated into four different forms by ion exchange chromatography on diethylaminoethyl-cellulose. 2. 2. Form A was purified 1300–1500 fold by an unusual procedure, the technique of ampholyte displacement, followed by chromatography on concanavalin A Sepharose. 3. 3. The enzyme catalyses the hydrolysis of both β- N-acetylglucosaminides and β- N-acetylgalactosaminides. 4. 4. The kinetic studies support the evidence of the association of both activities to a single protein, and at the same active site. 5. 5. A natural substrate, N, N′-diacetylchitobiose, is also hydrolyzed, but not ovalbumin. 6. 6. This enzyme may be considered as an exoglycosidase.

Ampholyte displacement and high pressure liquid chromatographic separation of the estrogen-responsive neurophysin from human plasma.

Estrogen-stimulated neurophysin (ESN) or oxytocin (OT)-neurophysin (Np) was measured in plasma of seven men before and after oral administration of 25 mg diethylstilbestrol (DES). Pre-DES levels of ESN averaged 0.93 +/- 0.3 (+/- SEM) ng/ml and increased to 29.8 +/- 6.5 and 25.4 +/- 5.1 ng/ml 24 and 48 h after DES treatment, respectively. To compare the estrogen-responsive Np in plasma with human OT-Np which is present in the posterior pituitary gland, the Np fraction of post-DES plasma was concentrated by double precipitation with ammonium sulfate and applied to ampholyte displacement and Sephadex G-75 columns. The Np fraction of this plasma extract contained ESN immunoreactivity (IR) but no nicotine-stimulated neurophysin-IR. ESN-IR of plasma and of an extract of human posterior pituitary eluted identically from a Sephadex G-75 column, indicating similar mol wt. The plasma extract containing ESN-IR eluted from the ampholyte displacement column at pH 4.3-4.2. No nicotine-stimulated Np (arginine vasopressin-Np)-IR was found in the plasma samples. ESN-IR in an extract of human posterior pituitary gland eluted from the ampholyte displacement column at the same pH as that of the ESN extracted from plasma. Peak ESN-IR-containing fractions from the ampholyte displacement were pooled, dialyzed, lyophilized, and reconstituted in appropriate carrier buffer for reverse phase high pressure liquid chromatography. The ESN-IR was resolved into two distinct ESN-IR peaks by high pressure liquid chromatography. Plasma and posterior pituitary gave identical pairs of peaks. Thus, the Np that is increased in human plasma in response to estrogen is identical to pituitary OT-Np, providing strong evidence that estrogen stimulates the human neurohypophysis.

Activator protein for the degradation of globotriaosylceramide by human alpha-galactosidase.

An activator protein which stimulates the degradation of globotriaosylceramide by human hepatic alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was isolated from human liver and purified some 1300-fold. The purified activator was heat stable up to 95 degrees C, its molecular weight was estimated at 20,000 by gel filtration. Ampholyte displacement chromatography resolved the activating factor into two fractions with isoelectric points at pH 4.6 and pH 4.8, respectively, with otherwise identical properties. The protein did not stimulate the hydrolysis of the water-soluble 4-methylumbelliferyl-alpha-galactoside.

Formation of a [ [formula omitted]]polypeptide hormone: Characterization and chemical quality control by ampholyte displacement radiochromatography

99 m Tc-complexes with the polypeptide hormone secretin in very low concentration were formed by the concentrated hydrochloric acid/vacuum evaporation/gentisic acid method. The 99 m Tc-secretin was characterized by a modified ampholyte radiochromatographic procedure, in addition to thin layer chromatography, gel chromatography and paper electrophoresis. High radiochemical purity and specific radioactivity were obtained. In vivo distribution studies were performed, and the conditions necessary for application of [ 99 m Tc]polypeptides as scintigraphic agents are discussed.

Heterogeneity of human pituitary neurophysins by ampholyte displacement chromatography

Heterogeneity of human pituitary neurophysins by ampholyte displacement chromatography

Substance P biosynthesis from high molecular weight proteins in rabbit spinal ganglia

Substance P (SP) has been shown to be one of the neuro-transmitters in primary afferent fibers. In this report, using original C4 gel, 35S-meth ionine-substan ce P (35S-SP) synthesized by incubating isolated rabbit spinal ganglia with 35S-methionine was analyzed by rever s ed-pha s e high performance liquid chromatography (HPLC) or thin layer chromatography (TLC). Incorporation of 35S-methi on in e into SP reached the maximum at 8 hr and was inhibited by 0.1 mM cyc1oheximide, suggesting that SP is synthesized by a conventional ribosomal process in spinal ganglia. We also reported the presence of high molecular weight proteins in spinal ganglia that may serve as putative precursors in the biosynthesis of SP. Thus, Sephadex G-75 chromatography of the extracts from rabbit spinal ganglia incubated with 35S-methionine gave high molecular weight proteins incorporating 35S-methionine. When these proteins were incubated with spinal ganglia homogenates and were subjected to Sephadex G-75, Sephadex G-15, TLC or HPLC, 35S-SP was identified. Further characterization of these proteins by Sephadex G-200, gel performance chromatography or ampholyte displacement chromatography revealed that SP might be synthesized via approx. 100,000 and/or 7,000 dalton basic proteins in perikarya in rabbit spinal ganglia.

Identification of proteins in pooled human follicular fluid which suppress follicular response to gonadotropins.

To evaluate the role of nonsteroidal, follicular fluid proteins in folliculogenesis, the 10-55% saturated ammonium sulfate fraction of pooled human follicular fluid was dialyzed against 0.025 M Tris/HCl (pH 7.5) using 10,000 molecular weight exclusion membranes, then passed through agarose immobilized textile dye. Activity was determined by test fraction inhibition of human menopausal gonadotropin (2 U human LH/FSH . day), induced ovarian weight, and serum estradiol increase in hypophysectomized, diethylstilbesterol-treated, 25-day-old female rats. Specific inhibition (89 +/- 6.8% SEM) of ovarian weight increase was found in the material (2 ml) eluted from an Orange A column with KCl (1.5 M, pH 6.8). Inhibitory activity of the Orange A-bound material, which eluted through a standardized Sephadex G-50 column, corresponded to a molecular weight of 13,000-25,000. Isoelectric focusing on a Sephadex G-15 support bed or ampholyte displacement chromatography of Orange A bound material demonstrated inhibitory activity at pH 3.5-4.5 and 6.5-7.0. No demonstrable activity was found in similar fractions eluted through a Concanavalin A-Sepharose 4B column before or after addition of alpha-methyl-D-mannoside (2 M, pH 7). When active fractions were heated (56 C, 1 h) or exposed to trypsin (10 mg/100 ml), activity was lost. When aliquots of the saturated ammonium sulfate-extracted, dialyzed, Orange A-bound eluent were separated by high performance liquid chromatography using gel exclusion columns, activity in the bioassay was recovered in the 13,000-35,000 molecular weight range. Although confirmatory data await further studies, it is tempting to speculate that this protein(s) may be an important inter- and/or intraovarian regulator of follicular response to gonadotropins.

An approach to ampholyte-displacement chromatography

An experimental approach to ampholyte-displacement chromatography is described in which a linear pH gradient is obtained by ampholyte displacement on DEAE-Sepharose CL-6B. The results show that a pH gradient is obtained whatever the initial pH of the ion exchanger. Ampholytes are bound selectively to the ion exchanger according to their respective p I values and to their charge in solution. This experimental approach to ampholyte-displacement chromatography demonstrates the applicability of this technique for the separation of closely related proteins.

Purification of mouse alpha-foetoprotein by ampholyte displacement chromatography.

Mouse alpha-foetoprotein (alphaFP) was isolated from H4 hepatoma tissue using Con A-Sepharose salt gradient ion exchange chromatography and ampholyte displacement chromatography, the latter being a new method for a sharp separation of proteins based on their different isoelectric point. The purity of the alphaFP was demonstrated by (i) the absence of contaminant on sodium dodecyl sulphate polyacrylamide gel electrophetic gels, (ii) Ouchterlony's immunodiffusion against monospecific antimouse alphaFP and the absence of precipitation against a polyvalent antinormal mouse serum, (iii) the production of a monospecific antiserum in a rabbit after injection of the purified antigen, and (iv) immunological unreactivity of the produced antiserum against normal hepatic tissue.

Two methods for the separation of human α-fetoprotein and albumin: (A) Affinity chromatography using blue sepharose CL-6B and (B) ampholyte displacement chromatography

Two methods for the separation of α-fetoprotein (AFB) and albumin using (A) affinity chromatography on Blue Sepharose CL-6B and (B) ampholyte displacement chromatography are described. The heterogeneity of immunoreactive human AFP is demonstrated by ampholyte displacement chromatography; possibly seven variants of AFP can be distinguished by this method.

Rat liver proteins capable of transferring phosphatidylethanolamine. Purification and transfer activity for other phospholipids and cholesterol.

Two proteins, one in a highly purified form, have been isolated from the soluble fraction of rat liver homogenate. These proteins accelerate the transfer of labeled phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, sphingomyelin, and cholesterol from liposomes to mitochondria or erythrocyte ghosts. The fraction obtained after ammonium sulfate precipitation, gel filtration on Sephadex G-75, ion-exchange chromatography on CM-cellulose, ampholyte displacement chromatography, and heat treatment exhibited an 876-fold increase in its phosphatidylethanolamine transfer activity as compared with the postmitochondrial supernatant adjusted to pH 5.1. Isoelectric focusing on polyacrylamide gels shows a single band between pH 8.6 and 9.0. The transfer activity is abolished by trypsin, but withstands 5-min heating at 90 degrees. After heat treatment, a single major band is seen on polyacrylamide gel electrophoresis followed by two minor ones. The molecular weight of the major band is 12,500, as determined by electrophoresis on 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. A molecular weight of 13,500 was calculated from molecular filtration through Sephadex G-50. The relative transfer activities toward the different phospholipids remain constant throughout the last three steps of the purification procedure in spite of the extensive change in the electrophoretic profile of the protein mixture. The cholesterol transfer activity remains unchanged after the final heat treatment as well. This indicates that all of the transfer activities are present in a single protein.

Ampholyte displacement chromatography — A new technique for the separation of proteins illustrated by the resolution of β-N-acetyl-D-hexosaminidase isoenzymes unresolvable by isoelectric focusing or conventional ion-exchange chromatography

The ‘B’ variant of pig epididymal β-N-acetyl-D-hexosaminidase has, for the first time, been resolved into two isoenzymes (designated Bα and Bβ). Like the ‘B’ variant of ox liver aryl sulphatase, the two β-N-acetyl-D-hexosaminidase isoenzymes could not be resolved by isoelectric focusing procedures, but could be separated by ion-exchange chromatography. Similar difficulties may be expected in the isoelectric focusing of other large, moderately basic proteins with closely similar isoelectric points and with a tendency to aggregate. The β-N-acetyl-D-hexosaminidase ‘B’ isoenzymes were successfully resolved using ampholyte displacement chromatography — a new technique in which the proteins are displaced from an ion exchange adsorbent using mixtures of amphoteric substances of closely similar isoelectric points.